Serum Albumin
Serum albumin is the most abundant protein in mammalian sera (40 g/I; approximately 0.7 mM in humans), and one of its functions is to bind molecules such as lipids and bilirubin (Peters T, Advances in Protein Chemistry 37:161, 1985). The half-life of serum albumin is directly proportional to the size of the animal, where for example human serum albumin (HSA) has a half-life of 19 days and rabbit serum albumin has a half-life of about 5 days (McCurdy T R et al, J Lab Clin Med 143:115, 2004). Human serum albumin is widely distributed throughout the body, in particular in the intestinal and blood compartments, where it is mainly involved in the maintenance of osmolarity. Structurally, albumins are single-chain proteins comprising three homologous domains and totaling 584 or 585 amino acids (Dugaiczyk L et al, Proc Natl Acad Sci USA 79:71, 1982). Albumins contain 17 disulfide bridges and a single reactive thiol, C34, but lack N-linked and 0-linked carbohydrate moieties (Peters, 1985, supra; Nicholson J P et al, Br J Anaesth 85:599, 2000). The lack of glycosylation simplifies recombinant expression of albumin. This property of albumin, together with the fact that its three-dimensional structure is known (He X M and Carter D C, Nature 358:209 1992), has made it an attractive candidate for use in recombinant fusion proteins. Such fusion proteins generally combine a therapeutic protein (which would be rapidly cleared from the body upon administration of the protein per se) and a plasma protein (which exhibits a natural slow clearance) in a single polypeptide chain (Sheffield W P, Curr Drug Targets Cardiovacs Haematol Disord 1:1, 2001). Such fusion proteins may provide clinical benefits in requiring less frequent injection and higher levels of therapeutic protein in vivo.
Fusion or Association with HSA Results in Increased In Vivo Half-Life of Proteins
Serum albumin is devoid of any enzymatic or immunological function and, thus, should not exhibit undesired side effects upon coupling to a bioactive polypeptide. Furthermore, HSA is a natural carrier involved in the endogenous transport and delivery of numerous natural as well as therapeutic molecules (Sellers E M and Koch-Weser M D, “Albumin Structure, Function and Uses”, eds Rosenoer V M et al, Pergamon, Oxford, p 159, 1977). Several strategies have been reported to either covalently couple proteins directly to serum albumins or to a peptide or protein that will allow in vivo association to serum albumins. Examples of the latter approach have been described e.g. in WO91/01743. This document describes inter alia the use of albumin binding peptides or proteins derived from streptococcal protein G for increasing the half-life of other proteins. The idea is to fuse the bacterially derived, albumin binding peptide/protein to a therapeutically interesting peptide/protein, which has been shown to have a rapid clearance in blood. The thus generated fusion protein binds to serum albumin in vivo, and benefits from its longer half-life, which increases the net half-life of the fused therapeutically interesting peptide/protein.
Association with HSA Results in Decreased Immunogenicity
In addition to the effect on the in vivo half-life of a biologically active protein, it has been proposed that the non-covalent association with albumin of a fusion between a biologically active protein and an albumin binding protein acts to reduce the immune response to the biologically active protein. Thus, in WO2005/097202, there is described the use of this principle to reduce or eliminate the immune response to a biologically active protein.
Albumin Binding Domains of Bacterial Receptor Proteins
Streptococcal protein G is a bi-functional receptor present on the surface of certain strains of streptococci and capable of binding to both IgG and serum albumin (Björck et al, Mol Immunol 24:1113, 1987). The structure is highly repetitive with several structurally and functionally different domains (Guss et al, EMBO J 5:1567, 1986), more precisely three Ig-binding motifs and three serum albumin binding domains (Olsson et al, Eur J Biochem 168:319, 1987). The structure of one of the three serum albumin binding domains has been determined, showing a three-helix bundle domain (Kraulis et al, FEBS Lett 378:190, 1996). This motif was named ABD (albumin binding domain) and is 46 amino acid residues in size. In the literature, it has subsequently also been designated G148-GA3.
Other bacterial albumin binding proteins than protein G from Streptococcus have also been identified, which contain domains similar to the albumin binding three-helix domains of protein G. Examples of such proteins are the PAB, PPL, MAG and ZAG proteins. Studies of structure and function of such albumin binding proteins have been carried out and reported e.g. by Johansson and co-workers (Johansson et al, J Mol Biol 266:859-865, 1997; Johansson et al, J Biol Chem 277:8114-8120, 2002), who introduced the designation “GA module” (protein G-related albumin binding module) for the three-helix protein domain responsible for albumin binding. Furthermore, Rozak et al have reported on the creation of artificial variants of the GA module, which were selected and studied with regard to different species specificity and stability (Rozak et al, Biochemistry 45:3263-3271, 2006; He et al, Protein Science 16:1490-1494, 2007). In the present disclosure, the terminology with regard to GA modules from different bacterial species established in the articles by Johansson et al and by Rozak et al will be followed.
Recently, variants of the G148-GA3 domain have been developed, with various optimized characteristics. Such variants are for example disclosed in PCT publications WO2009/016043 and WO2012/004384.
Clostripain
Clostripain, also known as endoproteinase Arg-C, is a two-chain proteinase that can be isolated from Clostridium histolyticum. Clostripain has been shown to have both proteolytic and amidase/esterase activity (Mitchell, et al (1968), J Biol Chem 243:4683-4692). Clostripain activity has been reported to be optimal in the pH range of 7.6-7.9.
Clostripain preferentially cleaves at the carboxyl group of arginine residues (Labrou et al (2004), Eur J Biochem 271(5):983-92; Keil (1992), “Specificity of proteolysis”, Springer-Verlag, pp 335), however the cleavage of lysyl bonds has also been reported. Clostripain has been shown to accept substrates containing Lys instead of Arg, however reaction rates are low in comparison to reactions with Arg containing substrates. For example, clostripain has been reported to cleave glucagon at Arg-Arg, Arg-Ala and the Lys-Tyr sites. The relative initial rates of hydrolysis of these three bonds are 1, 1/7 and 1/300 (Labouesses (1960), Bull Soc Chim Biol 42:1293-304).
Clostripain cleavage is frequently utilized in biomedical and biotechnological applications. Applications of clostripain cleavage include peptide mapping, sequence analysis, cell isolation, hydrolysis/condensation of amide bonds, and peptide synthesis.
Clostripain may for example be used in order to cleave off tags (such as His6, c-Myc, Flag and GST tags) used for protein purification and/or detection. Additionally, clostripain cleavage may be used during the production of amidated therapeutic polypeptides from a precursor polypeptide, whereby the resistance of the therapeutic polypeptide to proteolytic degradation by endogenous proteases upon administration to animal or human subjects is increased.
As evident from the different sections of this background description, the provision of polypeptide molecules with a high affinity for albumin and exhibiting high resistance to enzymatic cleavage, in particular by clostripain, is a key factor in the development of various biomedical, biotechnological and other applications, and there is therefore a need in the art of such polypeptide molecules.